The present invention concerns crystalline forms of polypeptides corresponding to the catalytic domain of receptor tyrosine kinases of the non-insulin receptor type. Such tyrosine kinases include receptors of a class that are not covalently cross-linked but are understood to undergo ligand-induced dimerization (such as the FGF-receptor), as well as cytoplasmic tyrosine-kinases. The invention also concerns methods for obtaining such crystals and to the high-resolution X-ray diffraction structures and atomic structure coordinates obtained therefrom. The crystals of the invention, and the atomic structure coordinates obtained therefrom, are useful for solving the crystal and solution structures of the tyrosine kinase domains and for identifying compounds that bind to domains of receptor and non-receptor tyrosine kinases.
Growth factors play important roles in the control of cell growth, differentiation, metabolism and oncogenesis. The signals generated by a growth factor are transduced across the cellular membrane by transmembrane receptors specific for the growth factor. The diverse biological effects of growth factors are mediated by a large family of cell surface transmembrane receptors with intrinsic protein tyrosine kinase (PTK) activity. The extracellular portion of receptor PTKs contain the binding site for its particular growth factor/ligand, whereas the tyrosine kinase activity resides in the cytoplasmic portion. Binding of a growth factor to the extracellular domain of this receptor results in autophosphorylation of specific tyrosine residues in the cytoplasmic domain. These phosphotyrosines either stimulate PTK activity or serve as binding sites for downstream signalling proteins containing Src-homology 2 (SH2) or phosphotyrosine binding (PTB) domains.
Eighteen classes or subfamilies of human receptor PTKs have been identified to date, including the insulin-receptor (IR), EGF-receptor, PDGF receptor and FGF-receptor. Ligand-induced dimerization of receptors such as the EGF, PDGF and FGF receptors is thought to be essential for activation. Growth factors, such as PDGF are dimeric molecules which, by themselves, are able to induce PDGF-receptor dimerization. However, FGFs are monomeric and are unable, by themselves; to induce receptor dimerization. Dimerization of FGF receptors is thought to be mediated by FGF in concert with heparin sulfate proteoglycans (soluble or cell surface bound).
In contrast to the EGF, PDGF and FGF receptors, which are monomeric and dimerize upon ligand binding, the insulin receptor exists as a xe2x80x9cdimer.xe2x80x9d In fact, the insulin receptor is a disulphide-linked xcex12xcex22 heterotetramer. Binding of insulin to the extracellular xcex1-chains is thought to cause a change within the quaternary structure of the receptor that results in autophosphorylation of specific tyrosines in the cytoplasmic portion of the xcex2 chains.
In an effort to elucidate the mechanisms underlying kinase activation, the crystal structure of such proteins is often sought to be determined. The crystal structures of several protein serine/threonine kinases have been reported: cyclic-AMP-dependent protein kinase (CAPK; Knighton et al., 1994); cyclin-dependent kinase 2 (CDK2; DeBondt et al., 1993); mitogen-activated protein kinase (MAPK; Zhang et al., 1994); and twitchin kinase (Hu et al., 1994). However, the crystalline structure of only one receptor tyrosine kinase has been determinedxe2x80x94the unphosphorylated apo form of the tyrosine kinase domain of the insulin receptor (Hubbard et al., 1994).
Despite these reports, the ability to obtain crystalline forms of the tyrosine kinase domains of non-insulin receptor tyrosine kinases; i.e., cytoplasmic tyrosine kinases and/or receptor tyrosine kinases that undergo ligand-mediated dimerization, has not been realized. A particularly illuminating example is the EGF receptor; to the Applicant""s knowledge, researchers armed with the knowledge of how to obtain crystals of the tyrosine kinase domains of both the insulin receptor and serine/threonine kinases have attempted to obtain crystals of the tyrosine kinase domain of EGF receptor without success.
The invention relates to crystalline forms of polypeptides corresponding to the catalytic domains of receptor tyrosine kinases of the non-insulin receptor type. Such tyrosine kinases include receptors that are not covalently cross-linked, but are believed to undergo ligand-induced dimerization, as well as cytoplasmic tyrosine kinases. The polypeptides of the invention include, but are not limited to, crystallized polypeptides corresponding to the native or mutated catalytic domain of tyrosine kinases (ie., the non-insulin receptor-type described above), derivative crystals (i.e., heavy atom derivatives), and co-crystals of the native or mutated catalytic domain in association with one or more compounds, including but not limited to cofactors, substrates, substrate analogs, inhibitors, allosteric effectors, etc., and preferably compounds that bind the catalytic site.
Preferably, the crystalline catalytic domains of the invention are of sufficient quality to provide for a determination of the three-dimensional X-ray diffraction structure of the crystalline polypeptide to a resolution of about 1.5 xc3x85 to about 2.5 xc3x85.
The invention is based, in part, on the Applicants"" discovery and elucidation of the sequence requirements for the successful crystallization of polypeptides corresponding to catalytic domains of receptor tyrosine kinases that are not covalently cross-linked and are believed to undergo ligand-induced dimerizationxe2x80x94a goal which heretofore remained elusive. In this regard, the Applicants have determined that at least about 20 amino acid residues (+/xe2x88x925 amino acid residues) upstream of the first glycine in the conserved glycine-rich region of the catalytic domain, and at least about 17 amino acid residues (+/xe2x88x925 amino acid residues) downstream of the conserved arginine located at the C-terminal boundary of the catalytic domain are required to engineer a polypeptide suitable for crystallization.
In those cases where the resulting polypeptide contains cysteine residues that interfere with crystallization, such cysteine residues can be substituted with an appropriate amino acid that does not readily form covalent bonds with other amino acid residues under crystallization conditions, e.g., such substitutions include, but are not limited to Ala, Ser, or Gly. Any cysteines located in a non-helical or non-xcex2-strand segment based on secondary structural assignments are candidates for replacement. Cysteines located in domains corresponding to the glycine-rich loop, the kinase insert, the juxtamembrane region or the activation loop are prime candidates for replacement. However, substitutions of cysteine residues that are conserved among the kinases should be avoided (e.g., substitutions of the highly conserved cysteine residues located at the C-terminus, positions 725 and 736 in FIG. 6A, should be avoided).
The invention is demonstrated by way of example, for the fibroblast growth factor (FGF) receptor-1 (FGF-R1). The examples demonstrate that the crystal structure of the tyrosine kinase domain of the FGF-R1 has been determined to 2.0 xc3x85 resolution; the crystal structure of the FGF-R1 catalytic domain in complex with an ATP analog is described to 2.3 xc3x85 resolution.
The crystalline catalytic domains are useful for elucidating the mechanism by which the receptor tyrosine kinases are activated by ligand-induced dimerization, and for the identification of compounds that bind to the catalytic domain.
As used herein, the following terms shall have the following meanings:
xe2x80x9cNative Tyrosine Kinase Domain or Native Catalytic Domain:xe2x80x9d As used herein, xe2x80x9cnative tyrosine kinase domainxe2x80x9d or xe2x80x9cnative catalytic domainxe2x80x9d refers to that portion or domain of a naturally occurring cytoplasmic tyrosine kinase or non-insulin receptor tyrosine kinase which possesses protein tyrosine kinase (xe2x80x9cPTKxe2x80x9d) activity as described in Mohammadi et al., 1991 and 1996.
xe2x80x9cHuman FLGK:xe2x80x9d As used herein, xe2x80x9chuman FLGKxe2x80x9d refers to the tyrosine kinase domain of human fibroblast growth factor receptor 1 (xe2x80x9cFGFR1xe2x80x9d) having the amino acid sequence of SEQ ID NO:1. Generally, human FLGK comprises a 310 amino acid residue fragment (residues 456 to 765) of human FGFR1.
xe2x80x9cFLGK:xe2x80x9d As used herein, xe2x80x9cFLGKxe2x80x9d refers to a mutant of human FLGK which is characterized by the amino acid sequence of SEQ ID NO:2. As compared to human FLGK, FLGK contains the following amino acid substitutions: Cys-488xe2x86x92Ala, Cys-584xe2x86x92Ser, Leu-457xe2x86x92Val, and has an additional five amino acid residues at the N-terminus (residues 1-5 of SEQ ID NO:2) (Ser-Ala:Ala-Gly-Thr).
xe2x80x9cMutant:xe2x80x9d As used herein, xe2x80x9cmutantxe2x80x9d refers to a polypeptide which is obtained by replacing at least one amino acid residue in a native tyrosine kinase domain with a different amino acid residue and/or by adding and/or deleting amino acid residues within the native polypeptide or at the N- and/or C-terminus of a polypeptide corresponding to a native tyrosine kinase domain and which has substantially the same three-dimensional structure as the native tyrosine kinase domain from which it is derived. By having substantially the same three-dimensional structure is meant having a set of atomic structure coordinates that have a root mean square deviation (r.m.s.d.) of less than or equal to about 2 xc3x85 when superimposed with the atomic structure coordinates of the native tyrosine kinase domain from which the mutant is derived when at least about 50% to 100% of the Cxcex1 atoms of the native tyrosine kinase are included in the superposition.
A mutant may have, but need not have, PTK activity. xe2x80x9cCrystal:xe2x80x9d As used herein, xe2x80x9ccrystalxe2x80x9d refers to a polypeptide-in crystalline form. The term xe2x80x9ccrystalxe2x80x9d includes native crystals, derivative crystals and co-crystals, as described herein.
xe2x80x9cNative Crystal:xe2x80x9d As used herein, xe2x80x9cnative crystalxe2x80x9drefers to a crystal wherein the polypeptide is substantially pure.
xe2x80x9cDerivative Crystal:xe2x80x9d As used herein, xe2x80x9cderivative crystalxe2x80x9d refers to a crystal wherein the polypeptide is in covalent association with one or more heavy-metal atoms.
xe2x80x9cCo-Crystal:xe2x80x9d As used herein, xe2x80x9cco-crystalxe2x80x9d refers to a crystal wherein the polypeptide is in association with one or more compounds. Such compounds include, by way of example and not limitation, cofactors, substrates, substrate analogues, inhibitors, allosteric effectors, etc. Preferred compounds include AMP-PCP and AMP-PNP.
xe2x80x9cCo-Complex:xe2x80x9d As used herein, xe2x80x9cco-complexxe2x80x9d refers to a polypeptide in association with one or more compounds as enumerated above.
xe2x80x9cAssociation:xe2x80x9d As used herein, xe2x80x9cassociationxe2x80x9d refers to a condition of proximity between xcex1-chemical entity or compound, or portions or fragments thereof, and tyrosine 35 kinase domain protein, or portions or fragments thereof. The association may be non-covalent, i.e., where the juxtaposition is energetically favored by, e.g., hydrogen-bonding, van der Waals, electrostatic or hydrophobic interactions, or it may be covalent.
xe2x80x9cActive Site:xe2x80x9d As used herein, xe2x80x9cactive sitexe2x80x9d refers to that site in tyrosine kinase domains where substrate peptide binding, ATP binding and cleavage occur. For human FLGK and FLGK, the active site comprises the catalytic loop, the activation loop-and the nucleotide binding loop and is characterized by at least amino acid residues Lys-514, Glu-531, Asp-623, Asn-628, the glycine-rich loop (amino acid residues 485-490), Asp-641 and Arg-627 (FIG. 3).
xe2x80x9cCatalytic Loon:xe2x80x9d As used herein, xe2x80x9ccatalytic loopxe2x80x9d refers to a loop in tyrosine kinase domains between xcex1E and xcex27 containing conserved amino acid residues that are believed to be important in the phosphotransfer reaction or enzymatic process. For human FLGK and FLGK the catalytic loop contains aspartic acid residue Asp-623, which acts as a catalytic base, and is characterized by at least amino acid residues 621 to 628 (FIG. 3).
xe2x80x9cActivation Loop:xe2x80x9d As used herein, xe2x80x9cactivation loopxe2x80x9d refers to a loop in tyrosine kinase domains between xcex28 and xcex1EF that is believed to act as a regulatory loop. For human FLGK and FLGK, the activation loop contains two autophosphorylation sites and is characterized by at least amino acid residues 640 to 663 (FIG. 3).
xe2x80x9cNucleotide Binding Loon or Glycine-Rich Loop:xe2x80x9d As used herein, xe2x80x9cnucleotide-binding loopxe2x80x9d or xe2x80x9cglycine-rich loopxe2x80x9d refers to a loop in tyrosine kinase domains between xcex21 and xcex22 which contains the protein kinase-conserved glycine-rich GXGXXG consensus sequence (where X is any amino acid). For human FLGK and FLGK the nucleotide binding loop is characterized by at least amino acid residues 485 to 490 (FIG. 3).
xe2x80x9cAutophosphorylation Site:xe2x80x9d As used herein, xe2x80x9cautophosphorylation sitexe2x80x9d refers to those tyrosine residues in tyrosine kinase domains that are phosphorylated by a tyrosine kinase domain. Human FLGK and FLGK have six (6) autophosphorylation sites: two in the activation loop (Tyr-653 and Tyr-654), one in the juxtamembrane region (Tyr-463), two in the kinase insert (Tyr-583 and Tyr-585) and one in the C-terminal lobe (Tyr-730) (Mohammadi et al., 1996).
xe2x80x9cJuxtamembrane Region:xe2x80x9d As used herein, xe2x80x9cjuxtamembrane regionxe2x80x9d refers to that portion of receptor tyrosine kinases located between the transmembrane helix and the tyrosine kinase domain. For human FGFR1 the juxtamembrane region is characterized by at least amino acid residues 398 to 470 (FIG. 6).
xe2x80x9cKinase Insert:xe2x80x9d As used herein, xe2x80x9ckinase insertxe2x80x9d refers a stretch of up to about one hundred amino acid residues which divides the tyrosine kinase domain of certain tyrosine kinases in two. For human FLGK and FLGK, the kinase insert is located between helices xcex1D and xcex1E (FIGS. 1 and 3), contains autophosphorylation sites Tyr-583 and Tyr-585, and is characterized by at least amino acid residues 575 to 596 (FIG. 3).
xe2x80x9cUnit Cell:xe2x80x9d As used herein, xe2x80x9cunit cellxe2x80x9d refers to the smallest and simplest volume element (i.e., parallelpiped-shaped block) of a crystal that is completely representative of the unit of pattern of the crystal. The dimensions of the unit cell are defined by six numbers: dimensions a, b and c and angles xcex1, xcex2 and xcex3 (Blundel et al., 1976). A crystal is an efficiently packed array of many unit cells.
xe2x80x9cMonoclinic Unit Cell:xe2x80x9d As used herein, xe2x80x9cmonoclinic unit cellxe2x80x9d refers to a unit cell wherein axe2x89xa0bxe2x89xa0c; xcex1=xcex3=90xc2x0; and xcex2 greater than 90xc2x0.
xe2x80x9cCrystal Lattice:xe2x80x9d As used herein, xe2x80x9ccrystal latticexe2x80x9d refers to the array of points defined by the vertices of packed unit cells.
xe2x80x9cSpace Group:xe2x80x9d As used herein, xe2x80x9cspace groupxe2x80x9d refers to the symmetry of a unit cell. In a space group designation (e.g., C2) the capital letter indicates the lattice type and the other symbols represent symmetry operations that can be carried out on the unit cell without changing its appearance.
xe2x80x9cAsymmetric Unit:xe2x80x9d As used herein, xe2x80x9casymmetric unitxe2x80x9d refers to the largest aggregate of molecules in the unit cell that possesses no symmetry elements, but that can be juxtaposed on other identical entities by symmetry operations.
xe2x80x9cCrystallopraphically-Related Dimer:xe2x80x9d As used herein, xe2x80x9ccrystallographically-related dimerxe2x80x9d refers to a dimer of two molecules wherein the symmetry axes or planes that relate the two molecules comprising the dimer coincide with the symmetry axes or planes of the crystal lattice.
xe2x80x9cNon-Crystallographically-Related Dimer:xe2x80x9d As used herein, xe2x80x9cnon-crystallographically-related dimerxe2x80x9d refers to a dimer of two molecules wherein the symmetry axes or planes that relate the two molecules comprising the dimer do not coincide with the symmetry axes or planes of the crystal lattice.
xe2x80x9cIsomorphous Replacement:xe2x80x9d As used-herein, xe2x80x9cisomorphous replacementxe2x80x9d refers to the method of using heavy-atom derivative crystals to obtain the phase information necessary to elucidate the three-dimensional structure of a native crystal (Blundel et al., 1976). The phrase xe2x80x9cheavy-atom derivatizationxe2x80x9d is synonymous with xe2x80x9cisomorphous replacement.xe2x80x9d
xe2x80x9cMolecular Replacement:xe2x80x9d As used herein, xe2x80x9cmolecular replacementxe2x80x9d refers to the method of calculating initial phases for a new crystal whose structure coordinates are unknown by orienting and positioning a molecule whose structure coordinates are known within the unit cell of the new crystal so as to best account for the observed diffraction pattern of the new crystal. Phases are then calculated from this model and combined with observed amplitudes to provide an approximate Fourier synthesis of the structure of the molecules comprising the new crystal. This, in turn, is subject to any of several methods of refinement to provide a final, accurate set of structure coordinates for the new crystal (Lattman, 1985; Rossman, 1972).
The amino acid notations used herein for the twenty genetically encoded L-amino acids are conventional and are as follows:
xe2x80x9cATP:xe2x80x9d As used herein, xe2x80x9cATPxe2x80x9d refers to adenosine triphosphate.
xe2x80x9cAMP-PCP:xe2x80x9d As used herein, xe2x80x9cAMP-PCPxe2x80x9d refers to adenylyl diphosphonate, a non-hydrolyzable analogue of ATP.
xe2x80x9cAMP-PNP:xe2x80x9d As used herein, xe2x80x9cAMP-PNPxe2x80x9d refers to adenylyl imidodiphosphate, a non-hydrolyzable analogue of ATP.
xe2x80x9cCxcex1:xe2x80x9d As used herein, xe2x80x9cCxcex1xe2x80x9d refers to the alpha carbon of an amino acid residue.